CD8+ cytotoxic T lymphocytes (CTL) and CD4+ T cells play crucial roles in host defense against human malignancies. However, it is also becoming clearer that neoplastic cells may evade cell-mediated immunity at multiple levels of the effector/target interaction which, consequently, may impact the metastatic process. We have continued to characterize immune effector mechanisms involved in antigen (Ag)-specific T cell-mediated cytotoxicity of human carcinoma, with emphasis on the role of Fas/Fas ligand (FasL) interactions. If it is a relevant process, it also raises the possibility that the failure of such solid tumor types to undergo Fas-mediated apoptosis may contribute to tumor escape. Thus, we have been exploring the hypothesis that tumor-cell resistance to Ag-specific T cell attack at the level of Fas/FasL engagement may underlie a unique tumor escape mechanism that may contribute to the progression of the neoplastic process. We have developed an in vitro model to compare the lytic sensitivity of a human primary colon carcinoma cell line (SW480) to its metastatic-derived isolate (SW620) to Ag-specific CTL, produced previously from normal or carcinoma-bearing individuals by peptide-based strategies. Here, we demonstrated that these CTL lysed the HLA-A2+ Ag-bearing SW480 and SW620 cell lines in vitro, although IFN-g treatment of the targets was necessary for regulation of the lytic outcome. Moreover, we found that the CTL mechanisms employed in lysis of the primary and metastatic cell lines were distinct. IFN-g pretreatment rendered SW480 cells sensitive to both Fas-dependent and Fas-independent (perforin) pathways, whereas SW620 cells displayed lytic susceptibility to Fas-independent mechanisms only. In addition to cytokine modulation, we have shown that treatment of SW480 cells with either 5-FU, CPT-11 or CDDP led to enhanced ICAM-1 and Fas expression, resulting in Ag-specific CTL-mediated lysis through Fas-dependent and Fas-independent pathways. In contrast, treatment of SW620 cells with either CPT-11 or CDDP, but not 5-FU, led to enhanced ICAM-1 expression, resulting in Ag-specific CTL killing via Fas-independent mechanisms only. These data support the hypotheses that: (a) resistance of certain carcinoma cells to the Fas pathway may represent unique tumor escape mechanisms in response to T cell attack; (b) IFN-g may help to enhance or restore a Fas-sensitive phenotype in certain carcinoma cells; and (c) certain anti-neoplastic agents may exhibit unique immunoregulatory properties that promote carcinoma cell death through engaging the cytotoxic capabilities of Ag-specific CTL. To further explore the potential association between Fas responsiveness and the malignant/metastatic phenotype, we selected for the outgrowth of Fas-insensitive SW480 cells by in vitro culture with either agonistic anti-Fas mAb or CD4+ T cell-derived sFasL (as a natural source of sFasL) and then compared the selected with the unselected tumor cells for differences in metastatic behavior in vivo, based on lymph node metastasis in an athymic mouse model. Overall, Fas-insensitive SW480 tumor cells could be derived, which exhibited enhanced malignant tendencies in vivo. Clearly, the destruction of carcinoma cells through cell contact-independent mechanisms, such as sFasL, may be important determinants against neoplastic cells that downregulate MHC/Ag complexes. However, the idea that sFasL may also "select" for carcinoma cells resistant to Fas-mediated apoptosis introduces the hypothesis that the antitumor immune response contributes, albeit inadvertently, to the progression of the neoplastic process. A second major effort of study involved the development and characterization of a mouse model based on immune recognition of tumor cells endogenously expressing tumor-specific Ag (TSA). The CMS4 tumor cell line (H-2d) is a chemically induced sarcoma established from BALB/c mice. Tumor-specific CD8+ CTL were produced, which recognized an H-2Ld-restricted nonamer peptide epitope derived from an envelop protein (gp70) of an endogenous murine leukemia retrovirus. This was based upon CTL cross-reactivity against syngeneic CT26 colon carcinoma cells previously characterized to express that gene product, as well as by cold target inhibition assays. The adoptive transfer of CMS4-reactive CTL in vivo resulted in potent antitumor activity against 3-day established pulmonary metastases. Our data also indicated that treatment of CMS4 cells with IFN-g plus TNF-a rendered them sensitive to Fas-mediated lysis ensuing engagement with agonistic anti-Fas mAb. The objectives of future studies include: (1) to further analyze the role of Fas-mediated lysis in the regulation of the anti-neoplastic response; (2) to further explore the potential mechanistic link between Fas and the malignant/metastatic phenotype; (3) to identify potential defects in tumor-cell death pathways which associate with immune resistance to a particular effector mechanism; and (4) to explore antitumor properties of Ag-specific CTL subtypes in vivo in murine models.